Influenza A viruses continue to be a public health concern due to the mortality and morbidity associated with seasonal (epidemic) and pandemic influenza. Although an influenza virus pandemic has not occurred during the last 40 years, most of the influenza virus experts agree that they will be a pandemic in the near future. However, we still have limited knowledge on the processes that govern influenza virus virulence and on the genesis of human pandemic influenza viruses. In order to gain some insights on this processes, we reconstructed the influenza virus strain that caused the 1918 influenza pandemic. During the previous period of support of this PPG, the multidisciplinary characterization of this strain performed by our research team revealed important features associated with its extreme virulence. Among these features, we recently determined that the viral genes PBl, HA and NA are the main responsible ones for enhanced virulence and replication. In Project 3, we will continue these studies by analyzing in more detail the molecular contributions to pathogenesis of the 1918 HA and NA proteins. This will be done by analyzing specific domains and properties of these proteins by biochemical techniques and also in the context of recombinant influenza viruses. The phenotype of the recombinant viruses in animal models will also be analyzed in collaboration with Core B. This analysis, in combination with the structural studies performed by our collaborators in Project 1, the systems biology studies conducted by Project 5, and the characterization of the PBl gene by Project 2, will reveal the molecular determinants of virulence of the 1918 virus. Human pandemic influenza viruses are known to be generated by reassortment processes between human and avian strains of influenza viruses and the acquisition of mammalian adaptive mutations. We will also conduct studies in vivo aimed to understand the temporal and spatial rules involved in the generation of reassortant influenza viruses. Finally, the adaptive mutations of the 1918 pandemic influenza virus that determine its replication in mammalian versus avian hosts will also be determined. Our proposed studies will then unravel new information on processes involved in virulence and host adaptation of influenza A viruses. RELEVANCE (See instructions): We will study two fundamental processes of the influenza virus biology of significant public health concern, molecular pathogenesis and host adaptive processes. Our studies then will offer new important information of interest for the adoption of measures that might mitigate the processes involved in the generation of pandemic influenza viruses, and the severity of influenza virus infections.